Journal:

Article Title: Identification of the Domains of UreR, an AraC-Like Transcriptional Regulator of the Urease Gene Cluster in Proteus mirabilis

doi: 10.1128/JB.183.15.4526-4535.2001

Figure Lengend Snippet: E. coli strains and plasmids used in this study

Article Snippet: After transformation, the pBAD vector containing the gene sequence encoding LexA 1–87 -Myc-His was purified using a Qiagen miniprep kit.

Techniques: Construct

Journal:

Article Title: Identification of the Domains of UreR, an AraC-Like Transcriptional Regulator of the Urease Gene Cluster in Proteus mirabilis

doi: 10.1128/JB.183.15.4526-4535.2001

Figure Lengend Snippet: Western blot analysis of fusion constructs. E. coli Top10 (A, B, C, and F) or E. coli JL1436 (D and E), transformed with vector or clones expressing UreR or chimeric proteins, was cultured in LB medium to mid-exponential phase and induced with 2% arabinose for 4 h (unless otherwise noted). Samples (10 μl) of each suspension were boiled for 5 min in gel sample buffer and electrophoresed on an SDS-polyacrylamide gel. Membranes were incubated with antiserum or monoclonal antibodies and developed with secondary antibodies conjugated to alkaline phosphotase (Sigma). (A) pBAD, UreR, UreR-Myc-His; 12.5% SDS-polyacrylamide gel. Membranes were reacted with mouse anti-His antibody (diluted 1:1,000) (Invitrogen). (B) pBAD, UreR, UreR164–293, or UreR164–293-Myc-His; 15% SDS-polyacrylamide gel. Membranes were reacted with mouse anti-His antibody (diluted 1:1,000) (Invitrogen). (C) pBAD, UreR, C/EBP302–350, C/EBP302–350-Myc-His; 15% SDS-polyacrylamide gel. Membranes were reacted with mouse anti-His antibody (diluted 1:1,000) (Invitrogen). (D) LexA, LexA1–87, LexA1–87-Myc-His; induced with 2 mM IPTG. Samples (2 μl) of each were electrophoresed on a 15% SDS-polyacrylamide gel. Membranes were hybridized with rabbit polyclonal anti-LexA antibody (diluted 1:5,000) (Invitrogen). (E) LexA, LexA1–87, UreR1–182-LexA1–87; induced with 2 mM IPTG. Samples (1.25 μl) of each, including strain only, were electrophoresed on a 12.5% SDS-polyacrylamide gel. Membranes were reacted with rabbit polyclonal anti-LexA antibody (diluted 1:5,000) (Invitrogen). (F) pBAD, UreR, UreR-Myc-His, UreR L147A-L148A, UreR L147A-L148A-Myc-His, UreR L147A-L148A-L158A, and UreR L147A-L148A-L158A-Myc-His; induced with 2% arabinose for 4 h. Samples (10 μl) of each were electrophoresed on a 12.5% SDS-polyacrylamide gel. Membranes were hybridized with mouse anti-Myc antibody (diluted 1:5,000) (Invitrogen).

Article Snippet: After transformation, the pBAD vector containing the gene sequence encoding LexA 1–87 -Myc-His was purified using a Qiagen miniprep kit.

Techniques: Western Blot, Construct, Transformation Assay, Plasmid Preparation, Clone Assay, Expressing, Cell Culture, Incubation

Journal:

Article Title: Identification of the Domains of UreR, an AraC-Like Transcriptional Regulator of the Urease Gene Cluster in Proteus mirabilis

doi: 10.1128/JB.183.15.4526-4535.2001

Figure Lengend Snippet: Gel mobility shift assay for interaction of UreR with the P. mirabilis pureD. A 60-bp double-stranded oligonucleotide was synthesized based on the sequence of pureD. Whole-cell extracts of E. coli Top10 transformed with either pBAD or vector expressing C/EBP302–350-UreR164–293 were obtained by inducing a 4-ml culture in mid-exponential phase with 2% arabinose for 4 h. Whole-cell extracts of E. coli Top10 expressing UreR and UreR-Myc-His were obtained by inducing a 4-ml culture in mid-exponential phase with 2% arabinose and 100 mM urea for 4 h. Bacterial suspensions were adjusted to the same OD, and 3 ml of each culture was harvested by centrifugation (10,000 × g, 5 min, 4°C). Bacteria were resuspended in 1 ml of TEN buffer and lysed in a French press (18,000 1b/in2). Binding reactions with the 60-bp pureD dsDNA fragment were carried out in the presence of 100 mM urea with various amounts of the extracts.

Article Snippet: After transformation, the pBAD vector containing the gene sequence encoding LexA 1–87 -Myc-His was purified using a Qiagen miniprep kit.

Techniques: Mobility Shift, Synthesized, Sequencing, Transformation Assay, Plasmid Preparation, Expressing, Centrifugation, Binding Assay